Organs and fluid spaces throughout the bodies of animals are lined by polarized epithelia that serve, in part, to modify the apical and basolateral fluid compartments. In order for these organs and fluid spaces to function, it is necessary that the apical and basolateral fluid compartments be kept physiologically separated, so as to maintain the modifications imparted by the epithelia. This physiological separation is maintained by the tight junction, classically known as the zonula occludens.
The tight junction is an unbroken intercellular junction formed by an anastomosing network of protein and lipid strands that apically circumscribes every luminal epithelial cell. This intercellular adhesion complex is a continuous molecular seal between all of the luminal epithelial cells and forms a selective barrier to paracellular, and thereby transepithelial, solute flux and ionic current (i.e., the gate function). The tight junction is also believed to maintain the distinct lipid and protein composition of the apical and basolateral cellular membranes (i.e., the fence function). The gate and fence functions of the tight junction thus polarize the unit epithelial cell and the epithelium itself at the same physical plane. Occludin and the claudins help to form, and are localized within, tight junction strands where they participate in homophilic and/or heterophilic interactions between adjacent cells. As schematically illustrated in FIG. 1A, occludin and the claudins are tetraspanin proteins, having intracellular N and C termini, two extracellular loops, and four transepithelial domains, and have been shown to be involved in establishing and maintaining the physiological properties of the tight junction.
In addition to physiological barrier roles, the tight junction regulates many aspects of intracellular behavior. For example, the tight junction has been shown to be involved in the cell cycle arrest attendant on contact inhibition. Tight junction disruption induces epithelial to mesenchymal transition, increases cellular motility, produces overgrowth of cultured cells, and increases tumorigenicity of cells transplanted into animals. Several tight junction proteins regulate transcription. Tight junction formation is also involved in the inter-related phenomena of development of cellular polarity and epithelial differentiation.
The physiological barrier functions as well as the regulatory activities of the tight junction must be maintained in epithelia whose cellular populations are undergoing constant turnover throughout the life of the organism. In this process individual epithelial cells are extruded apically and undergo apoptosis in a manner that does not alter the electrical resistance or tracer flux across the epithelium.
Higher levels of apoptosis induced in epithelia during experiments or during pathological states have been shown to alter barrier properties. Disruption of occludin has been shown in a number of studies to disrupt the physiological and structural properties of the tight junction. A 19 amino acid second extracellular loop sequence peptide mimic of occludin impeded recovery of tight junction structure following a short period of incubation with a calcium-free solution (the calcium switch) in T84 intestinal epithelial cells.
Isolated patches of cells throughout treated monolayers showed punctate, intracellular distributions of the tight junction proteins ZO-1, occludin, claudin-1, and JAM-A. A similar peptide was used to treat the EPH4 mammary epithelial cell line leading to punctate, intracellular, non-tight junctional distribution of occludin in isolated patches of cells throughout treated monolayers. Another similar peptide was shown to disrupt barrier function in a sertoli cell line. This same peptide shut down spermatogenesis and decreased testicular weight when injected into the testicular lumen of rats. The relationship between occludin disruption and cellular survival has not been widely studied. However, disruption of several types of cellular adhesion proteins; integrins, cadherins, and connexins has been reported to stimulate apoptosis. Interestingly, epithelial cell lines derived from the occludin knockout mouse showed decreased survival signaling and increased apoptotic rates.
Although most tumors are devoid of tight junctions, many tight junction proteins, particularly claudins 3, 4 and 7 are found at high levels in tumors of epithelial origin where their function and localization are presently unknown.
Although tight junction protein modulators are known, these are not without any problems. Therefore, there is a continuing need for other tight junction protein modulators.